In vitro immune-enhancing effects of Platycodon grandiflorum combined with Salvia plebeian via MAPK and NF-κB signaling in RAW264.7 cells

The immune-enhancing activity of the combination of Platycodon grandiflorum and Salvia plebeian extracts (PGSP) was evaluated through macrophage activation using RAW264.7 cells. PGSP (250–1000 μg/mL) showed a higher release of NO in a dose-dependent manner. The results showed that PGSP could significantly stimulate the production of PGE2, COX-2, TNF-α, IL-1β, and IL-6 in RAW264.7 cells and promote iNOS, COX-2, TNF-α, IL-1β, IL-4, and IL-6 mRNA expression. Western blot analysis demonstrated that the protein expression of iNOS and COX-2 and the phosphorylation of ERK, JNK, p38, and NF-κB p65 were greatly increased in PGSP-treated cells. PGSP also promoted the phagocytic activity of RAW264.7 cells. All these results indicated that PGSP might activate macrophages through MAPK and NF-κB signaling pathways. Taken together, PGSP may be considered to have immune-enhancing activity and might be used as a potential immune-enhancing agent.

Although individual immunostimulatory effects have been demonstrated, the mixture of P. grandiflorum and S. plebeia has not been previously studied, and the effects of the combination of these plants remain unclear.A number of studies demonstrated that the combination of plant materials enhanced immunity both in vivo and in vitro, showing a more powerful bioactive effect than single plant species alone [16,[34][35][36][37]. Therefore, the objective of this study was to determine if the effects on immune function are enhanced by treating murine macrophages with a mixture of P. grandiflorum and S. plebeian (PGSP).

Preparation of PGSP
A mixture of PGSP was supplied after manufacturing by FD FARM Co.,Ltd.(Incheon, Korea).Briefly, dried P. grandiflorum and S. plebeian at a ratio of 1:1 (v/v) were mixed and extracted by adding distilled water at 100˚C ± 5 for 6-8 h.The water extract was concentrated using a vacuum concentrator until the solid content reached a brix of 50 ± 5.0 to obtain PGSP extract.PGSP was stored at -4˚C and used in this study.The ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to identify and quantify platycodin D and platycoside E in PSGP were presented in Supplemental Material.The UPLC-MS/MS analysis indicated that the PGSP contained 4 saponins, including platycoside D1 (1.11 ± 0.02 mg/g), platycoside D2 (1.73 ± 0.04 mg/g), platycoside E1 (2.40 ± 0.09 mg/g), and platycoside E2 (1.04 ± 0.09 mg/g).

Cell culture and treatment
Mouse macrophage cell lines were obtained from the Korean Cell Line Bank (Korean Cell Line Research Foundation, Seoul, Korea) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Welgene, Korea) and 1% penicillin/streptomycin (P/S, Welgene, Korea) in humidified incubators containing 5% CO 2 .Various concentrations of PGSP were diluted in RPMI-1640 medium without phenol red, supplemented with 1% FBS and 1% P/S.Concentrations of 250, 500, 750, and 1000 μg/mL of PGSP were added to the cells for 24 h.Lipopolysaccharide (LPS, 1 μg/mL) was used as the positive control.After incubation, the immunological effects were assessed in subsequent experiments.

Cell viability analysis
The EZ-Cytox Cell Viability Assay Kit (Daeil Labservice, Seoul, Korea) was used to determine cell viability.RAW264.7 cells (1 × 10 6 cells/mL) were cultured with samples (250-1000 μg/mL) and incubated for 24 h.The supernatants were removed, and 100 μL of a water-soluble tetrazolium salt (WST) solution was added to each well.The PGSP-treated cells were incubated at 37˚C for 1 h.The absorbance was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA).

Determination of NO production
Griess reagent (Sigma-Aldrich, St. Louis, MO, USA) was used for the detection of NO production in macrophages.The cells (1 × 10 6 cells/mL) were treated with PGSP for 24 h before the culture supernatants (100 μL) was transferred into each well of a 96-well plate.An equal volume (100 μL) of Griess reagent (0.1% N-1-napthylethylenediamine dihydrochloride in distilled water and 1% sulfanilamide in 5% phosphoric acid) was added to and incubated for 10 min at room temperature.Finally, the absorbance at 540 nm was measured using a microplate reader.

Real-time qPCR analysis
Tri reagent 1 (Molecular Research Center, Inc., Cincinnati, OH, USA) was used to extract total RNA from LPS-and PGSP-treated and untreated RAW264.7 cells at a density of 1 × 10 6 cells/mL, according to the manufacturer's protocol.cDNA was synthesized using 1000 ng of RNA in the following steps: 25˚C for 10 min, 37˚C for 120 min, and 85˚C for 5 min, using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA).To quantify the mRNA levels of gene expression, real-time qPCR analysis was performed using TB Green 1 Premix Ex Taq™ II (Takara Bio Inc., Shiga, Japan) that mixed a final concentration of 0.4 μM of a specific primer pairs, 1 × ROX Reference Dye, and 5 ng of cDNA templates, and then carried out on a QuantStudio™ 3 FlexReal-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA).The reaction steps were as follows: 95˚C for 30 s, 40 cycles of 95˚C for 5 s, and 60˚C for 34 s.Then, 95˚C for 30 s, 60˚C for 1 min, and 95˚C for 15 s were added at the end of the cycles.All reactions was carried out in triplicate.The relative expression of the target genes was calculated and normalized to that of β-actin as an internal control.A list sequences of mouse primers are provided in Table 1.

Assay of macrophage phagocytosis
The PGSP-treated cells at a density of 2 × 10 6 cells/mL were harvested and washed with icecold phosphate-buffered saline (PBS).After that, a solution of FITC-dextran (Sigma-Aldrich, USA) was added to PGSP-treated cells for 1 h at 37˚C.The reaction was stopped by adding 1 mL of ice-cold PBS, and the cells were washed three times with 1% paraformaldehyde.Phagocytic uptake was analyzed using the CytoFLEX Flow Cytometer (Beckman Coulter, Inc., USA).

Analysis of western blotting
The PGSP-treated cells (2 × 10 6 cells/mL) were extracted in 120 μL of lysis buffer containing radioimmunoprecipitation assay (RIPA) buffer, 0.5 mM ethylenediaminetetraacetic acid (EDTA), and an inhibitor cocktail (protease and phosphatase).The cell lysates were incubated at 4˚C for 30 min and then centrifuged at 13,000 rpm for 20 min.The protein concentration in the supernatant was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA) and estimated using the standard curve, as directed by the manufacturer.Subsequently, equal volumes of proteins were resolved on sodium dodecyl sulfate-polyacrylamide gels and then transferred to polyvinylidene membranes.The membranes were blocked for 1 h in 5% skim milk in 1 × Tris-buffered saline containing 1% Tween-20 (1 × TBST).After incubation, the membranes were washed with 1 × TBST and incubated overnight with the primary antibody (1:2000).Then, the membranes were washed and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L) (1:2000).The protein bands were detected by Pierce 1 ECL Plus Western Blotting Substrate and visualized with the ChemiDoc XRS + imaging system (Bio-Rad, Hercules, CA, USA).

Data analysis
The data were analyzed using SPSS version 23.0 software (SPSS, Inc., Chicago, IL, USA) and presented as the mean ± standard deviation (SD).Each value is the mean of three separate experiments (n = 3).The significance was analyzed using a one-way analysis of variance followed by Duncan's new multiple range test.P-values of < 0.05 were considered statistically significant.

Effect of PGSP on cell viability
As shown in Fig 1, the cell viability after treatment with PGSP concentrations of 250, 500, 750, and 1000μg/mL was 101.12 ± 1.35%, 98.07 ± 0.59%, 98.48 ± 0.71%, and 100.59 ± 0.77%, respectively, but statistically different from the RPMI controls (p < 0.05).Neither PGSP nor LPS were cytotoxic to RAW264.7 cells within the tested concentration range, suggesting that these doses of PGSP could be used in further experiments without causing cytotoxicity.

Effects of PGSP on the NO production
After 24 h of incubation with PGSP, the production of NO in RAW264.7 macrophages was measured.As shown in Fig 2A, PGSP showed a significant stimulatory effect on NO production in a dose-dependent manner, which was 81.19 ± 1.30% at the highest concentration (1000 μg/mL).The amount of NO produced by PGSP was comparable to that of the positive control, LPS (1 μg/mL), indicating that it exerted strong stimulant activity.

Effect of PGSP on iNOS and COX-2 mRNA and protein expression
Real-time qPCR and western blot analyses were performed on RAW264.7 macrophages to examine whether the induction of immunomodulators like NO and PGE 2 occurred due to mRNA and protein expression of iNOS and COX-2.As shown in Fig 4A and 4B, the mRNA expression levels of iNOS and COX-2 were significantly enhanced in a concentration-dependent manner after treatment of the cells with PGSP for 24 h.PGSP concentration-dependently induced the expression of iNOS and COX-2 protein (Fig 4C).Additionally, PGSP induced iNOS and COX-2 expression at a lower level than that in LPS-treated cells but stronger than that in RPMI-treated cells.Therefore, PGSP may regulate pro-inflammatory mediators, such as iNOS and COX-2.

Effects of PGSP on phagocytic activity
A fluorescein isothiocyanate (FITC)-dextran uptake assay was performed to measure the effects of PGSP or LPS on macrophage phagocytosis.As shown in Fig 5, the results revealed that LPS could increase macrophage phagocytic activity by 68.77 ± 0.07%, which was displayed by higher FITC-fluorescence intensity than the untreated control group.Flow cytometry revealed that the percentage of phagocytosis increased to 41.02 ± 2.27%, 54.69 ± 2.70%, 62.72 ± 2.90%, and 69.22 ± 1.59% in cells treated with 250, 500, 750, and 1000 μg/mL, respectively.

Effects of PGSP on NF-κB and MAPK activation
To investigate the molecular mechanisms underlying the immune-enhancing effects of PGSP, the phosphorylation of NF-κB subunit p65, JNK, ERK, and p-38 MAPK was evaluated in RAW264.7 cells by western blotting, and the results are shown in Fig 6 .A dose-dependent increase in NF-κB and MAPK activation was observed by treatment with PGSP compared to RPMI.PGSP induced NF-κB-p65, ERK1/2, p38, and JNK phosphorylation, indicating NF-κB and MAPK activation.LPS also strongly increased NF-κB and MAPK expression levels (Fig 6 ).

Discussion
Previous pharmacological studies on P. grandiflorum mainly examined its immune-enhancing effects in vitro and in vivo [15,18].P. grandiflorum extracts primarily contained platycodin D and exhibited immuno-enhancing effects both in vitro and in vivo [16,17,38].Treatment of RAW264.7 cells with the combination of fermented P. grandiflorum and soybean extract or vitamin C showed immune responses greater than treatment with fermented P. grandiflorum alone [16].S. plebeian extracts were also used in these studies to modulate immunity in the forced swimming exercise-induced model [30].S. plebeian extracts contained higher quantities of flavonoids, such as luteolin, luteoloside, nepetin, nepitrin, hispidulin, homoplantagenin, and eupatorine [39], which have anti-inflammatory effects.These flavonoid compounds displayed immunomodulatory effects in various cells [40].However, the action of P. grandiflorum mixed with S. plebeian has not been reported.In this study, we investigated the immunostimulatory effects of a mixture of P. grandiflorum and S. plebeia (PGSP) in a macrophage cell line and the mechanism through which PGSP enhanced inflammatory responses.
Macrophages are known to be involved in multiple biological functions, such as inflammation control and immune enhancement, by releasing pro-inflammatory cytokines and inflammatory factors [41,42].The phagocytic activity of macrophages is a key indicator of macrophage activation in non-specific immune responses [43].The results of this study indicated that PGSP significantly improved macrophage phagocytosis, consistent with previously reported results [43][44][45][46].The results showed that PGSP activated macrophages to improve immunity.
The signal transduction molecules NO and PGE 2 play important roles in inflammation.Fermented P. grandiflorum extract enhanced NO production at the highest concentration (500 μg/mL), but the effect was only slightly statistically significant compared to the control group [15].In this study, PGSP significantly stimulated NO production at a dose range of 250-1000 μg/mL.Similarly, mixtures of Sasa quelpaertensis Nakai and Ficus erecta var.sieboldin effectively stimulated NO production by RAW264.7 cells in a dose range of 10-1000 μg/ mL [37].The inflammatory response involves the enzymes iNOS and COX-2, which regulate the production of NO and PGE 2 .The results of this study showed that PGSP increased NO and PGE 2 secretion and upregulated iNOS and COX-2 expression.PGSP also enhanced immunity by upregulating iNOS and COX-2 protein expression in macrophage cells.Ko et al. (2012) [1] investigated the immunomodulatory effects of Abelmoschus esculentus extracts on RAW2647 cells by regulating the production of TNF-α, IL-1β, IL-6, NO, and PGE 2 , as well as the expression of iNOS and COX-2 [1].The combination of Panax ginseng and Scrophularia buergeriana increased phagocytosis, cytokine, and NO production and enhanced iNOS expression via NF-κB activation in RAW264.7 cells and immune responses in splenocytes [46].
Macrophage activation is associated with the generation of immunomodulators through MAPK and NF-κB pathways [1,2].P. grandiflorum polysaccharides were shown to activate MAPK and AP-1 in macrophages [49].Our study also demonstrated that PGSP could dosedependently induce the activation of signaling molecules, such as NF-κB and MAPKs, in RAW264.7 cells, indicating that these molecules regulated a variety of cellular functions, including cell survival, proliferation, and apoptosis [50].These results suggested that PGSP strongly enhanced macrophage cellular immunity by stimulating the phosphorylation of ERK, JNK, p38 MAPK, and NF-κB p65.Althaea rosea extracts were found to exhibit immunomodulatory activity by increasing NO, cytokines, and mediator secretion by activating NF-κB and MAPK proteins in macrophages [48].Therefore, the present results suggest that PGSP enhanced immune responses by stimulating macrophages through NF-κB and MAPK signaling.

Conclusion
PGSP showed strong immunoenhancing activity in macrophages by increasing the secretion of NO, PGE 2 , cytokines, mediators, and phagocytosis, as well as increasing the expression of immune-related genes and the activation of NF-κB and MAPK signaling pathways.Thus, PGSP could act as a potent immunomodulator derived from P. grandiflorum and S. plebeian and could also be developed as a functional food or supplementary diet ingredient for health.

Fig 1 .
Fig 1.Effect of PGSP on the viability of RAW264.7 macrophages.Cell viability was determined by the EZ-Cytox Cell Viability Assay Kit.All values are presented as the mean ± SD of three independent experiments (n = 3).A different letter (p < 0.05) reveals statistically significant differences within treatments.https://doi.org/10.1371/journal.pone.0297512.g001